Received July 6, 2015; Revision received July 21, 2015
We report on the effects of high light irradiance (480 µmol
quanta/(m2·s)) and salinity (160 and 200 g/liter
NaCl) on culture growth as well as on cell lipid pigment and fatty acid
(FA) composition in three novel strains of halophile microalga from the
genus Dunaliella. Based on the ITS1–5.8S rRNA–ITS2
sequence and on the capability of accumulation of secondary (uncoupled
from the photosynthetic apparatus) β-carotene, the strains
Dunaliella sp. BS1 and BS2 were identified as D. salina
and Dunaliella sp. R5 as D. viridis. Under conditions
optimal for growth, chlorophylls and primary carotenoids (mainly
lutein) dominated the pigment profile of all investigated strains. The
main FA were represented by unsaturated C18 FA typical of thylakoid
membrane structural lipids. In all studied cells, stressors caused a
decline in chlorophylls and an increase in unsaturated C16 and C18 FA
associated with reserve lipids. The carotenogenic species D.
salina demonstrated 10-fold increase in carotenoids accompanied by
a decline in lutein and a drastic increase in β-carotene (up to
75% of total carotenoids). In D. viridis, only 1.5-fold increase
in carotenoid content took place, the ratio of major carotenoids
remaining essentially unchanged. The role of the carotenogenic response
in mechanisms of protection against photooxidative damage is discussed
in view of halophile microalgae stress tolerance and application of the
new Dunaliella strains for biotechnological production of
β-carotene.
KEY WORDS: carotenoids, chlorophylls, fatty acids, molecular
identification, photoacclimation, salt tolerance, stress

Halophile single-celled algae (microalgae) from the genus
Dunaliella (Chlorophyta, Chlorophyceae) are the main
phytoplankton component and primary producer in continental hypersaline
water bodies characterized by volatile and often unfavorable
environmental conditions [1, 2]. Among the halophile microalgae, Dunaliella
salina draws considerable attention for its capability of
significant (up to 10% of cell dry weight) accumulation of secondary
(i.e. structurally and functionally uncoupled from the photosynthetic
apparatus) β-carotene [3-5]. This feature renders D. salina the most
important microalgal source of biotechnologically produced natural
β-carotene [2, 6].

The capability of secondary carotenogenesis, among other factors,
determines the high tolerance of certain representatives of the genus
Dunaliella to extremely high salinities, irradiances, and
temperatures as well as to mineral nutrient deficiency [7-9]. In microalgae including
Dunaliella, the stress-induced secondary carotenogenesis is
accompanied by a reduction in the photosynthetic apparatus and
transition of the lipid metabolism to production of reserve neutral
lipids [10-12]. Studies of
the coordinated biosynthesis of secondary carotenoids (Car) and reserve
lipids are of considerable interest for revealing stress-tolerance
mechanisms in single-celled phototrophic organisms. One should also
emphasize that a pronounced carotenogenic response to stresses is
encountered only in selected representatives of the genus
Dunaliella, being a species-specific trait of D.
salina.

In consideration of the forgoing, we turned our attention to newly
described Dunaliella strains with different carotenogenic
capacity isolated from hypersaline water bodies of the Russian
Federation and preliminarily identified as D. salina [13]. Here, we report on the updated taxonomic status
of these Dunaliella strains, the induction of their
carotenogenic response, and the changes in fatty acid (FA) profile of
their cell lipids induced by high irradiance and salinity levels. The
new findings are discussed in view of halophile microalgae tolerance to
photooxidative damage and possible biotechnological application of
these Dunaliella strains.

MATERIALS AND METHODS

Microalgae and cultivation conditions. Algological monocultures
isolated from shallow ephemeral lagoons near the delta of
Bol’shaya Smorogda River flowing into Elton Lake (Volgograd
Region, Russia) – strains Dunaliella sp. BS1 and BS2, as
well as from hypersaline Razval Lake (Orenburg Region, Russia) –
Dunaliella sp. R5, were used in this work. The latter strain was
preliminarily identified as D. salina and deposited into the
IPPAS collection (Timiryazev Institute of Plant Physiology, Russian
Academy of Sciences) under ID IPPAS D-232 [13].

Genomic DNA isolation, sequencing, and sequence analysis. The
cells (2-5 mg dry weight) were pelleted by centrifugation,
freeze–thawed three times, and incubated for 1 h in 300 µl
of citrate-phosphate buffer (pH 5.0) containing 1 µM EDTA and 2%
SDS at 40°C. After addition of 1 M NaCl, the samples were left
overnight for protein salting, then DNA was extracted by a standard
phenol–chloroform method [15]. The DNA
preparation quality was checked by electrophoresis in 1.5% agarose gel
stained with 0.2 µg/ml ethidium bromide.

The following oligonucleotide primers were used to amplify the target
DNA fragment (~600 bp) containing the internal transcribed spacers ITS1
and ITS2 (fragment) as well as the 5.8S rRNA gene from the nuclear
ribosomal gene cluster (ITS1–5.8S rRNA–ITS2):
3′-GCCTGCCTACCCAGTTGCG-5′ and
5′-GAACCTGCGGAAGGATCATTG-3′ [16]. PCR
amplification was carried out by a Mastercycler Gradient amplifier
(Eppendorf, Germany) using Taq-polymerase (Evrogen, Russia) according
to the following program: 95°C, 3 min – initial denaturation;
95°C, 20 s; 55°C, 30 s; and 72°C, 35 s (30 cycles);
72°C, 5 min – final elongation. The PCR product was purified
using the Cleanup Standard kit (Evrogen) according to the
manufacturer’s manual and sequenced using a 3730 DNA Analyzer
automatic sequencer (Applied Biosystems, USA).

Homologous sequences were searched in the NCBI GenBank (nr/nt) database
using the online BLAST [17] service. The sequences
were analyzed using MEGA 6.06 software [18];
multiple alignment was performed with MUSCLE software [19] using the default parameters (see Supplement to
this paper on the site of the journal http://protein.bio.msu.ru/biokhimiya).

Pigment and total cell lipid FA extraction and assay.
Photosynthetic pigments were extracted from the microalgal cells with
chloroform–methanol mixture (2 : 1 v/v), chlorophylls (Chl)
a and b as well as total Car were assayed in the
chloroform fraction of the extract spectrophotometrically [20], and FA profile of the total cell lipids was
resolved using GC-MS [14].

All experiments were carried out in triplicate. Average values with
corresponding standard errors are shown unless stated otherwise.

RESULTS AND DISCUSSION

Studies of secondary carotenogenesis in microalgae are important for
deciphering the mechanisms of stress tolerance of phototrophic
organisms as well as for the biotechnological production of value-added
carotenoid pigments [8, 10].
Therefore, potentially carotenogenic organisms such as the new
Dunaliella representatives studied in this work are of
considerable interest. Based on cytological and morphological criteria,
all the studied strains were preliminarily identified as D.
salina. However, significant physiological and biochemical
differences between the strains (e.g. in their carotenogenic potential,
see below) found in this work poised the need for a revision of their
taxonomical status. Towards this end, the genomic DNA fragments
including partial sequences of ITS1 and ITS2 as well as complete 5.8S
rRNA gene were amplified and sequenced, and the corresponding GenBank
IDs are KT013269 (strain BS1), KT013270 (BS2), and KT013271 (strain R5
IPPAS D-232). Comparison of the ITS1–5.8S rRNA–ITS2
sequences with those of known microalgae revealed high (95-99%)
homology with representatives of the genus Dunaliella
(Volvocales, Dunaliellaceae). The strain Dunaliella sp. R5
clustered (Fig. S1 in Supplement) with strains
forming the so-called “viridis clade III”. Accordingly, the
strain Dunaliella sp. R5 could be identified as D.
viridis [21]. The strains Dunaliella
sp. BS1 and BS2 clustered together with the strains belonging to the
“salina clade III” [21], thereby
allowing to identify them as D. salina.

Since the carotenogenic response was of special interest in the context
of the present research, we followed the changes in cell number and
morphology at high irradiance (480 µmol PAR
quanta/(m2·s)) and NaCl level (160 or 200 g/liter) in
the cultivation medium – the most efficient inducers of secondary
carotenoid accumulation in Dunaliella [2-5].

The strains identified as belonging to different species exhibited
different trends in cell number changes during cultivation (figure).
Thus, the D. salina BS1 and BS2 cultures (figure, panel (a)) had
a low growth rate during the first nine days of cultivation. After this
period, the growth rate increased sharply, leading to a 3-4-fold
increase in cell number. At the 16th (or, in some cases, at the 11th)
day of cultivation, the culture growth slowed and a sharp decline in
cell number took place. We did not detect a profound effect of NaCl
concentration in the medium the growth rate in these strains. Cell
death was observed in the D. viridis R5 cultures initiated at
the same cell density and grown under similar conditions as the
cultures of D. salina (not shown). Although an increase in the
initial cell density of the D. viridis R5 cultures prevented the
cell death, no detectable culture growth was observed at 160 g/liter
NaCl (figure, panel (b), curve 1). Still, there was a sizeable
decline in the cell number in the cultures grown at elevated salinity
level (200 g/liter NaCl; figure, panel (b), curve 2).

The cultivation of the microalgae under conditions inducing
carotenogenesis was accompanied by species-specific changes in cell
morphology. In the case of D. viridis R5, the cell coloration
changed from green to yellow, and colorless inclusions appeared in the
cytoplasm (Fig. S2 in the Supplement). The cells
of D. salina BS1 and BS2 increased their size, assumed a
roundish form, and accumulated large globules in the cytoplasm. The
cells of these strains acquired first yellowish and then uniformly
orange-red of brown-red coloration (Fig. S2)
typical of the carotenogenic Dunaliella species [1-4].

The pre-cultures of D. viridis R5 grown under the optimal
conditions had similar pigment composition, high Chl content and,
according to the results of the HPLC analysis, relatively low
proportion of β-carotene in total Car (Table 1), typical of primary Car of green microalgae and
higher plants grown under optimal conditions [22].
The cells of D. salina BS1 and BS2 pre-cultures were
characterized by a 15-20% higher proportion of β-carotene in total
Car (Table 1).

Table 1. Pigment composition of studied
halophile microalgae from the genus Dunaliella on the culture
initiation (0 day) and after 23 days of cultivation at 480 µmol
photons/(m2·s) in the presence of different NaCl
concentrations in the medium
* Standard error (<5% of the average) is not shown.
** Traces.

A decline in total Chl along with increase in Chl a/b
ratio was normally observed in the course of cultivation under
stressful conditions. An increase in absolute Car content as well as
Car/Chl ratio also comprised a common trend, but the studied strains
differed dramatically in the magnitude of the changes. Thus,
approximately 1.5-fold increase in Car content and Car/Chl ratio was
recorded in D. viridis R5, whereas in D. salina BS1 and
BS2 these parameters increased more than by one order of magnitude
(Table 1). At the same time, the Car profile of
D. viridis R5 remained essentially unchanged. By contrast, the
Car composition of D. salina BS1 and BS2 was characterized by an
increase in the proportion of β-carotene up to 75-88% of total
Car, mainly at the expense of a corresponding decline in lutein (Table
1). The decline in absolute content of lutein
serving mainly the function of light harvesting is in accord with the
observed decline in Chl. These phenomena might manifest a typical for
microalgae response to stresses involving the reduction of the light
harvesting antenna in order to mitigate the risk of photooxidative
damage, which can be high under stress [23].

The processes described above were accompanied by a characteristic
dynamics of total cell lipid FA profile (Table 2). In all cases studied, the major FA were myristic
(14:0, 0.8-12.8% of total FA), palmitic (16:0, 22.4-38.3%), palmitoleic
(Δ9-16:1, 1.8-11.9%), hexadecatetraenoic (Δ4,7,10,13-16:4,
0.8-13.6%), stearic (18:0, 0.7-11.4%), and, in the case of D.
viridis, α-linolenic (Δ9,12,15-18:3, 16.8-36.9%) acid
as well. The minor FA included pentadecanoic (15:0), margaric (17:0),
arachidic (20:0), and cis-vaccenic (Δ7-18:1) acids
(<0.3% of total FA). The proportion of myristic acid in total cell
lipids of D. salina BS1 grown at 160 g/liter NaCl declined
2.3-fold, whereas in the other strains this FA increased considerably
(2.6-12.8 times). At the same time, D. salina BS1 and D.
viridis displayed an increase in palmitic and stearic FA,
suggesting vigorous biosynthesis of myristate, which is converted to
palmitate and further elongated to stearate. The latter two saturated
FA are known as the major FA of reserve lipids – triacylglycerols
[24]. Similar changes of FA composition were
recorded in strain BS2 as well as during cultivation of D.
salina in the presence of 200 g/liter NaCl. Noteworthy, trienoic FA
(represented mainly by α-linolenic acid associated predominantly
with the thylakoid membrane glycolipids [24])
declined considerbly in D. viridis. In cells grown in the
presence of 160 g/liter NaCl, this process occurred on the background
of an increase in dienoic FA, whereas more intense salinity stress (200
g/liter NaCl) promoted an increase in monoenoic FA. These changes in
the FA composition might reflect the operation of two stress
acclimation mechanisms: reduction of the thylakoid membrane and
induction of reserve lipid biosynthesis providing a sink for the
excessive photosynthates appearing as a result of slowing of cell
division [25]. This is a plausible explanation of
a stronger correlation between accumulation of β-carotene and
increase in palmitate or oleate (but not with the increase in total FA)
in cells of carotenogenic Dunaliella species [10]. The observed features of FA composition are also
consistent with the currently accepted central role of FA biosynthesis
in the buildup of carotenogenic response. FA biosynthesis is the source
of the building blocks of triacylglycerols, the major constituents of
plastoglobuli harboring the secondary β-carotene accumulating
under stress [8, 12].

Table 2. Changes in total cell lipid fatty
acid composition of the studied Dunaliella strains grown for
nine days at 480 µmol photons/(m2·s) in medium
containing 160 g/liter NaCl
* Percentage of total FA (standard error <5% of average).
** Sums of all isomers of corresponding FA are specified.
*** Not detected.

Summarizing the data available in the literature and obtained in the
present work, we conclude that the capability of secondary
carotenogenesis is a key determinant of the strategy of acclimation to
harsh environmental conditions. Thus, species of Dunaliella
lacking the ability to accumulate high amounts of secondary carotenoids
(e.g. D. viridis) respond to high light and salinity stresses
mostly by decline in Chl, reduction of the thylakoid membrane apparatus
[26, 27], and accumulation of
osmoprotectants such as glycerol [2]. Carotenogenic
Dunaliella species such as D. salina, apart from a
decline in Chl, accumulate β-carotene, enhancing cell protection
against the stresses by optical screening [28] and
a possible local antioxidant effect [29].
Furthermore, the biosynthesis of FA and neutral lipids coordintaed with
the accumulation of secondary Car might provide an additional sink for
excesive photsynthates, thereby lowering the risk of photooxidative
damage, which is especially high under the combination stress (high
light intensity and high salinty) [26].

Judging from the growth curves, the carotenogenic species D.
salina more efficiently acclimated to the stresses simulated under
our experimental conditions that the non-carotenogenic D.
viridis; the adverse effects of the elevated salinity were much
less expressed in D. salina than in D. viridis (figure).
Collectively, a considerably higher stress tolerance together with the
capability of β-carotene accumulation suggest that the new D.
salina strains BS1 and BS2 have considerable potential for
biotechnological production of this value-added pigment.

The authors are grateful to Dr. A. O. Plotnikov for his assistance in
obtaining of the microalgal cultures and Dr. T. A. Fedorenko for her
help with DNA isolation.

Funding by the Russian Scientific Found is greatly appreciated (project
No. 14-50-00029).